Journal: Advanced Science

Article Title: SerpinA3 is an Endogenous TGF‐β Receptor Antagonist that Attenuates Cardiac Fibroblast Activation and Fibrotic Remodeling

doi: 10.1002/advs.77379

Figure Lengend Snippet: SerpinA3K is downregulated in the pressure overload–induced failing heart. C57BL/6J mice underwent TAC surgery for 8 weeks; the control group underwent Sham surgery. (A) Left ventricular tissues from Sham and TAC mice were analyzed by tandem mass tag‐based quantitative proteomic analysis ( n = 3). The volcano map shows differentially expressed proteins between the 2 groups. (B) Gene Ontology analysis of the differentially expressed genes between the 2 groups. (C) The heatmap illustrates the expression differences of proteins related to serine‐type endopeptidase inhibitor activity between the 2 groups. (D) Protein levels of SerpinA3K, Collagen I, Periostin (POSTN), and Connective tissue growth factor (CCN2) in the left ventricle of mice detected by western blot analysis. (E) Quantification of SerpinA3K, Collagen I, POSTN, and CCN2 in (D). (F) Representative confocal microscopy images of immunofluorescence staining for SerpinA3K (green) and DAPI (blue) of the left ventricle. Scale bar: 50 µm. (G) Quantification of SerpinA3K fluorescence intensity in (F). (H) Protein levels of SerpinA3K in plasma of mice detected by western blot analysis. (I) Quantification of SerpinA3K in (H). Data are mean ± SEM, n = 6 mice per group, unpaired 2‐tail t test.

Article Snippet: Analysis involved the primary antibodies including GAPDH (5174T; Cell Signaling Technology, Danvers, MA, USA), P‐Smad2/3 (8828S; Cell Signaling Technology, Danvers, MA, USA), Smad2/3 (8685S; Cell Signaling Technology, Danvers, MA, USA), SerpinA3K (55480‐1‐AP; Proteintech, Wuhan, CN), SerpinA3 (sc‐69983; Santa cruz, Dallas, TX, USA), Collagen I (A1352; ABclonal, Wuhan, CN), POSTN (A14556; ABclonal, Wuhan, CN), CCN2 (25474‐1‐AP; Proteintech, Wuhan, CN), TGFR‐1 (sc‐518018; Santa cruz, Dallas, TX, USA), TGFR‐2 (sc‐17791; Santa cruz, Dallas, TX, USA), FLAG (14793S; Cell Signaling Technology, Danvers, MA, USA), HA (3724S; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Control, Expressing, Activity Assay, Western Blot, Confocal Microscopy, Immunofluorescence, Staining, Fluorescence, Clinical Proteomics

Journal: Advanced Science

Article Title: SerpinA3 is an Endogenous TGF‐β Receptor Antagonist that Attenuates Cardiac Fibroblast Activation and Fibrotic Remodeling

doi: 10.1002/advs.77379

Figure Lengend Snippet: SerpinA3 inhibits cardiac fibroblast activation by blocking TGF‐β signaling. (A) The signature genes for each subcluster are represented by dot plots. The color and size of the dots indicate the relative average expression level in each population and the percentage of cells expressing the gene, respectively. (B) Dot plot representation of GO terms that are derived from differentially expressed genes in the Saline and SerpinA3 groups. (C) C57BL/6J mice underwent Sham or TAC surgery, followed by intraperitoneal injection of recombinant human SerpinA3 protein (100ug/kg) every 3 days for 8 weeks. Protein levels of p‐Smad2/3, t‐Smad2/3, Collagen I, POSTN, and CCN2 in the left ventricle of mice were detected by western blot analysis. (D) Quantification of p‐Smad2/3/t‐Smad2/3, Collagen I, POSTN, and CCN2 in (C). (E) Mouse primary CFs (mPCFs) were pretreated with different concentrations of recombinant human SerpinA3 protein for 30 min, followed by 2 h of TGFβ1 (10 ng/mL) stimulation. Protein levels of p‐Smad2/3 and t‐Smad2/3 were detected by western blot analysis. (F) Quantification of p‐Smad2/3/t‐Smad2/3 in (E) ( n = 5 independent experiments). (G) mPCFs were treated with recombinant human SerpinA3 protein (100ug/mL) for 30 min before treatment with TGFβ1 (10 ng/mL) for 2 h. Immunofluorescence staining of total‐Smad2/3 (green) and DAPI (blue). Scale bars: 20 µm. (H) The ratio of the nuclear/ cytoplasmic fluorescence intensities of total‐SMAD2/3 in (G). (I) mPCFs migration was assessed by Transwell migration assay. (J) mPCFs proliferation was assessed by CCK‐8 assay. (K) The contractility of mPCFs was assessed by Gel contraction assay. (L) Quantification of the shrinkage area of the gel in (K). (M) Representative confocal microscopy images of immunofluorescence stained for a‐SMA (green), phalloidin (red), and DAPI (blue). Scale bars: 20 µm. (N) Quantification of α‐SMA fluorescence intensity in (M). Data are mean ± SEM, n = 6 independent experiments, 2‐way ANOVA with Bonferroni post‐test.

Article Snippet: Analysis involved the primary antibodies including GAPDH (5174T; Cell Signaling Technology, Danvers, MA, USA), P‐Smad2/3 (8828S; Cell Signaling Technology, Danvers, MA, USA), Smad2/3 (8685S; Cell Signaling Technology, Danvers, MA, USA), SerpinA3K (55480‐1‐AP; Proteintech, Wuhan, CN), SerpinA3 (sc‐69983; Santa cruz, Dallas, TX, USA), Collagen I (A1352; ABclonal, Wuhan, CN), POSTN (A14556; ABclonal, Wuhan, CN), CCN2 (25474‐1‐AP; Proteintech, Wuhan, CN), TGFR‐1 (sc‐518018; Santa cruz, Dallas, TX, USA), TGFR‐2 (sc‐17791; Santa cruz, Dallas, TX, USA), FLAG (14793S; Cell Signaling Technology, Danvers, MA, USA), HA (3724S; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Activation Assay, Blocking Assay, Expressing, Derivative Assay, Saline, Injection, Recombinant, Western Blot, Immunofluorescence, Staining, Fluorescence, Migration, Transwell Migration Assay, CCK-8 Assay, Collagen Gel Contraction Assay, Confocal Microscopy

Journal: Advanced Science

Article Title: SerpinA3 is an Endogenous TGF‐β Receptor Antagonist that Attenuates Cardiac Fibroblast Activation and Fibrotic Remodeling

doi: 10.1002/advs.77379

Figure Lengend Snippet: Myofibroblast‐targeted AAV‐mediated SerpinA3 overexpression ameliorates pressure overload–induced cardiac remodeling. (A) C57BL/6J mice were injected with AAV9‐ postn ‐Ctrl and AAV9‐ postn ‐SerpinA3 via the tail vein and underwent Sham or TAC surgery for 8 weeks. (B) M‐mode echocardiographic images of 4 groups mice. (C, D) Echocardiographic analysis of left ventricular ejection fraction (EF) and fractional shortening (FS) in each group. (E, F) Ratio of heart weight (HW) to body weight (BW), and ratio of heart weight (HW) to tibial length (TL) in each group. (G) Representative images of Gated wheat germ agglutinin (WGA) staining of the left ventricle. Scale bar: 50 µm. (H) Quantification of the cross‐sectional area of the left ventricular cardiomyocytes in (G). (I) Representative images of Masson staining and Sirius red staining of the left ventricle. Scale bar: 200 µm. (J, K) Quantification of fibrotic area and collagen content of the left ventricular in (I). (L) Representative confocal microscopy images of immunofluorescence staining for a‐SMA (green) and vimentin (VIME, fibroblast marker) (red). Scale bar: 50 µm. (M) Quantification of co‐localization of a‐SMA and VIME fluorescence intensity in (L). (N) Protein levels of p‐SMAD2/3, t‐SMAD2/3, Collagen I, POSTN, and CCN2 in the left ventricle of mice detected by western blot analysis. (O) Quantification of p‐SMAD2/3/t‐Smad2/3, Collagen I, POSTN, and CCN2 in (N). Data are mean ± SEM, n = 6 mice per group, 2‐way ANOVA with Bonferroni post‐test.

Article Snippet: Analysis involved the primary antibodies including GAPDH (5174T; Cell Signaling Technology, Danvers, MA, USA), P‐Smad2/3 (8828S; Cell Signaling Technology, Danvers, MA, USA), Smad2/3 (8685S; Cell Signaling Technology, Danvers, MA, USA), SerpinA3K (55480‐1‐AP; Proteintech, Wuhan, CN), SerpinA3 (sc‐69983; Santa cruz, Dallas, TX, USA), Collagen I (A1352; ABclonal, Wuhan, CN), POSTN (A14556; ABclonal, Wuhan, CN), CCN2 (25474‐1‐AP; Proteintech, Wuhan, CN), TGFR‐1 (sc‐518018; Santa cruz, Dallas, TX, USA), TGFR‐2 (sc‐17791; Santa cruz, Dallas, TX, USA), FLAG (14793S; Cell Signaling Technology, Danvers, MA, USA), HA (3724S; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Over Expression, Injection, Staining, Confocal Microscopy, Immunofluorescence, Marker, Fluorescence, Western Blot

Journal: International Journal of General Medicine

Article Title: A POSTN + CAF/APOE + TAM Axis is Associated with Tumor Progression and Potential Immunotherapy Resistance in Laryngeal Squamous Cell Carcinoma

doi: 10.2147/IJGM.S583478

Figure Lengend Snippet: Intercellular communication between POSTN + CAFs and APOE + TAMs in LSCC. ( A ) Global ligand–receptor interaction networks reveal strong bidirectional communication between POSTN + CAFs and APOE + TAMs. ( B ) Outgoing signaling patterns from POSTN + CAFs. ( C ) Outgoing signaling patterns from APOE + TAMs. (In figure A-C, the numbers on the edges indicate the amount of inferred significant intercellular interactions between cell populations.) ( D ) Bubble plots highlight significant ligand–receptor pairs mediating communication from POSTN + CAFs. ( E ) GO enrichment indicates chemotaxis and apoptotic signaling modulation as major biological processes (from POSTN + CAFs to APOE + TAMs). ( F ) KEGG analysis identifies cytokine-cytokine receptor interactions, complement/coagulation as dominant pathways (from POSTN + CAFs to APOE + TAMs). ( G ) Bubble plots highlight significant ligand–receptor pairs mediating communication from APOE + TAMs. ( H ) GO enrichment indicates chemotaxis and apoptotic signaling modulation as major biological processes (from APOE + TAMs to POSTN + CAFs). ( I ) KEGG analysis identifies proteoglycans in cancer, cytokine-cytokine receptor interaction as dominant pathways (from APOE + TAMs to POSTN + CAFs).

Article Snippet: Sections were then incubated with primary antibodies against POSTN (Epizyme Biotech, catalog number: R014951) and APOE (Epizyme Biotech, catalog number: R013930), followed by FITC- and Cy3-conjugated secondary antibodies.

Techniques: Chemotaxis Assay, Coagulation

Journal: International Journal of General Medicine

Article Title: A POSTN + CAF/APOE + TAM Axis is Associated with Tumor Progression and Potential Immunotherapy Resistance in Laryngeal Squamous Cell Carcinoma

doi: 10.2147/IJGM.S583478

Figure Lengend Snippet: Correlation and spatial colocalization between POSTN + CAFs and APOE + TAMs in LSCC. ( A – C ) Scatter plots showing the positive correlation between POSTN + CAF abundance and APOE + TAM abundance across multiple cohorts, including TCGA-HNSC, GSE41613 , and GSE65858 . Shaded areas represent 95% confidence intervals, and the correlation was evaluated using Spearman’s test. ( D ) Representative multiplex immunofluorescence staining images of LSCC tissues showing colocalization of POSTN (red) and APOE (green) proteins. DAPI (blue) marks cell nuclei.

Article Snippet: Sections were then incubated with primary antibodies against POSTN (Epizyme Biotech, catalog number: R014951) and APOE (Epizyme Biotech, catalog number: R013930), followed by FITC- and Cy3-conjugated secondary antibodies.

Techniques: Multiplex Assay, Immunofluorescence, Staining

Journal: International Journal of General Medicine

Article Title: A POSTN + CAF/APOE + TAM Axis is Associated with Tumor Progression and Potential Immunotherapy Resistance in Laryngeal Squamous Cell Carcinoma

doi: 10.2147/IJGM.S583478

Figure Lengend Snippet: Protein–protein interaction between POSTN and APOE. ( A ) Structural model of the POSTN-APOE complex; the inset highlights key hydrogen-bonding and hydrophobic interactions. ( B and C ) Electrostatic potential maps of POSTN ( B ) and APOE ( C ), showing charge complementarity at the interface. ( D ) A 2D interaction diagram illustrating hydrogen bonds and hydrophobic contacts between key residues. Symbols used: red lines for hydrogen bonds, blue circles for non-ligand bonds, and pink arcs for hydrophobic contacts.

Article Snippet: Sections were then incubated with primary antibodies against POSTN (Epizyme Biotech, catalog number: R014951) and APOE (Epizyme Biotech, catalog number: R013930), followed by FITC- and Cy3-conjugated secondary antibodies.

Techniques:

Journal: International Journal of General Medicine

Article Title: A POSTN + CAF/APOE + TAM Axis is Associated with Tumor Progression and Potential Immunotherapy Resistance in Laryngeal Squamous Cell Carcinoma

doi: 10.2147/IJGM.S583478

Figure Lengend Snippet: Prognostic and functional implications of POSTN + CAFs and APOE + TAMs co-enrichment. ( A ) Kaplan-Meier survival analysis shows the worst outcomes in the POSTN + CAFs high /APOE + TAMs high group. ( B ) GO analysis reveals enrichment in extracellular matrix organization and collagen regulation in the POSTN + CAFs high /APOE + TAMs high group. ( C ) GSEA indicates activation of EMT, antigen presentation, neuroactive ligand–receptor signaling, and TGF-β pathways in the POSTN + CAFs high /APOE + TAMs high group.

Article Snippet: Sections were then incubated with primary antibodies against POSTN (Epizyme Biotech, catalog number: R014951) and APOE (Epizyme Biotech, catalog number: R013930), followed by FITC- and Cy3-conjugated secondary antibodies.

Techniques: Functional Assay, Activation Assay, Immunopeptidomics

Journal: International Journal of General Medicine

Article Title: A POSTN + CAF/APOE + TAM Axis is Associated with Tumor Progression and Potential Immunotherapy Resistance in Laryngeal Squamous Cell Carcinoma

doi: 10.2147/IJGM.S583478

Figure Lengend Snippet: Association of the POSTN + CAFs/APOE + TAMs axis with immune suppression and ICI resistance. ( A ) TME immune cell infiltration across LSCC clinical subgroups quantified by five deconvolution algorithms. ( B ) High POSTN + CAFs/APOE + TAMs enrichment LSCC shows increased stromal and immune scores but reduced tumor purity, as well as TIDE scores with a tendency to be higher. ( C – E ) The IMvigor210 cohort validates poorer survival in POSTN + CAFs high /APOE + TAMs high patients. ( F ) Treatment-response comparison shows a higher proportion of PD/SD in the high-risk group ( χ² -test).

Article Snippet: Sections were then incubated with primary antibodies against POSTN (Epizyme Biotech, catalog number: R014951) and APOE (Epizyme Biotech, catalog number: R013930), followed by FITC- and Cy3-conjugated secondary antibodies.

Techniques: Comparison